Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.

Cytogenetic mapping of lambda gt10 lacZ sequences in the transgenic mouse strain 40.6 (Muta Mouse).

The transgenic mouse strain 40.6 (Muta Mouse) was developed for the detection of gene mutations induced in vivo. Strain 40.6 was constructed by microinjecting the shuttle vector lambda gt10 lacZ into the male pronucleus of a single cell embryo resulting from a CD2 (i.e. BALB/c x DBA/2)F1 x CD2F1 cross. Approximately 40 concatenated copies of the shuttle vector were integrated per haploid genome. The resulting mice were bred to disomy for the insert for use in mutagenicity studies. Ultimately, it is hoped that transgenic rodent model systems such as this one will play an important regulatory role in hazard identification. Despite the increasing use of this strain in toxicological studies, relatively little is known about the site of integration of the target gene into the mouse genome. In this study, fluorescence in situ hybridization and DAPI chromosome banding were combined to determine the location of the transgenic element in the mouse genome. The results indicate that the lambda sequences containing the lacZ gene are located in the B region of mouse chromosome 3. No other major chromosomal rearrangements were evident in the genome of this mouse strain.

In vivo rodent erythrocyte micronucleus assay.

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (474) for easy reference. Introduction, purpose, scope, relevance, application and limits of test. The analysis of immature erythrocytes in either bone marrow or peripheral blood is equally acceptable for those species in which the spleen does not remove micronucleated erythrocytes. In the mouse, mature erythrocytes are also an acceptable cell population for micronucleus analysis when the exposure duration exceeds 4 weeks. Test substances. Organic solvents such as DMSO are not recommended. Freshly prepared solutions or suspensions should be used unless stability data demonstrate the acceptability of storage. Vegetable oils are acceptable as solvents or vehicles. Suspension of the test chemicals is acceptable for p.o. or i.p. administration but not for i.v. injection. The use of any unusual solvent should be justified. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and square root of 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

Report from the working group on the in vivo mammalian bone marrow chromosomal aberration test.

The following summary represents a consensus of the working group, except where noted. The goal of this working group was to identify the minimal requirements needed to conduct a scientifically valid and practical in vivo chromosomal aberration assay. For easy reference, the items discussed are listed in the order in which they appear in OECD guideline 475. Specific disagreement with the current and/or proposed OECD guideline is presented in the text. Introduction, purpose, scope, relevance, application, and limits of test: This test would not be appropriate in situations where there was sufficient evidence to indicate that the test article or reactive metabolites could not reach the bone marrow. Test substances: Solid and liquid test substances should be dissolved, if possible, in water or isotonic saline. If insoluble in water/saline, the test substance should be dissolved or homogeneously suspended in an appropriate vehicle (e.g., vegetable oil). A suspension was not considered suitable for an intravenous injection. The use of dimethyl sulfoxide as an organic solvent was not recommended. The use of any uncommonly used solvent/vehicle should be justified. Freshly prepared solutions or suspensions of the test substance should be employed unless stability data demonstrate the acceptability of storage. Selection of species: Any commonly used rodent species was deemed acceptable but rats or mice were preferred, with no strain preference. Number and sex: A consensus could not be reached as to the requirement for both sexes versus one sex in this assay. It was suggested that a single sex should be used unless pharmacokinetic and/or toxicity data indicated a difference in metabolism and/or sensitivity between males and females. The size of the experiment (i.e., number of cells per animal, number of animals per treatment group) should be based on statistical considerations. Lacking a formal analysis, it was agreed that at least 100 metaphase cells should be scored per animal while at least five animals of any one sex should be evaluated per treatment group. Recently, a formal analysis of the numbers of cells to score per animal and numbers of animals to score per treatment group was conducted at a workshop on statistics for in vivo mutagenicity tests (Adler et al., 1994). The conclusion of this workshop was that, based on a type I error of 0.05 and a power of 80% to detect at least a doubling in the control frequency, the minimal number of cells to score per animal was 200 and the minimal number of animals to score per sex per treatment group was four.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenic activity of 3 industrial chemicals in a battery of in vitro and in vivo tests.

3 chemicals were selected for mutagenicity testing from a priority list, based on production volume and available mutagenicity data. Propargyl alcohol (PA), 2-nitroaniline (2NA), and 5-methyl-1H-benzo-triazole (MBT) were selected for testing using the approach recommended in the Health Protection Branch Genotoxicity Guidelines. The battery of tests included the Salmonella/mammalian microsome mutation assay, the in vitro chromosomal aberration assay, and the bone-marrow micronucleus assay. The results indicate that 2 of the 3 chemicals, PA and 2NA, were clastogenic in vitro. Both PA and 2NA induced chromosomal aberrations in CHO cells in vitro with and without metabolic activation, while none induced reverse mutations detectable with the Salmonella/mammalian microsome assay. Because PA and 2NA were found to be in vitro clastogens, they also were tested in the mouse bone-marrow micronucleus assay. 2NA induced a small increase in micronuclei in males but not females. PA did not induce an increase in micronuclei.

Suitability of human chromosome-specific DNA libraries for mutagenicity studies in Macaca fascicularis.

The development and use of chromosome-specific DNA probes to label entire human chromosomes has been an important advance in molecular cytogenetics. Fluorescence in situ hybridization using whole chromosome-specific DNA probes has been used to study both numerical and structural chromosomal aberrations in many human cell types. It would be useful if this technology could be applied to other animal species. However, whole chromosome-specific DNA probes have been reported for only human chromosomes. In this study, experiments were conducted to determine whether human probes could be used to label chromosomes of the cynomolgus monkey, Macaca fascicularis. The results demonstrate that some human DNA probes are suitable for the study of chromosomal aberrations in the monkey. Monkey chromosomes 1 and 4 labelled with human DNA probes had a strong, chromosome-specific labelling pattern. The probe for human chromosome 21 labelled the short arm on the monkey chromosome 2 and the probe for human chromosome 2 could not be detected on any of the monkey chromosomes.

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate.

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.

Mutagenic activity of p-toluenesulfonhydrazide.

Toluenesulfonhydrazide (TSH) is a high volume production chemical for which there is relatively little toxicological data. In this study, the mutagenic activity of TSH was determined in the Salmonella/mammalian microsome assay and the in vitro chromosomal aberration assay using Chinese hamster ovary cells. TSH induced gene mutations both with and without metabolic activation in the Salmonella/mammalian microsome assay but that it did not induce chromosomal aberrations in Chinese hamster ovary cells. The results of this study indicate that TSH is an in vitro mutagen and should be assessed for in vivo mutagenicity.

The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells.

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.

The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The protocol recommended for the micronucleus assay in mammalian bone marrow has been revised and simplified. The number of sample times has been reduced to one or two, depending upon the dosing protocol. The minimum number of cells to be scored per treatment group has been increased to 20,000 to increase the ability of the assay to detect a doubling of the control micronucleus frequency. Use of both male and female animals is recommended. Scoring of micronuclei in polychromatic erythrocytes of peripheral blood is included as a variation of the bone marrow assay. Published data on chemicals tested by the micronucleus assay have been reviewed and are summarized.

The Auger electron dosimetry of indium-111 in mammalian cells in vitro.

Most of the radionuclides used in the formulation of radiopharmaceuticals emit Auger electrons when they undergo radioactive decay. The release of these low-energy electrons at extracellular sites produces little direct damage to intracellular structures. However, many radiopharmaceuticals, or their metabolites, can be transported into the cell where the Auger electrons have the potential to damage nearby intracellular macromolecules, including DNA. In this preliminary study, chromosome damage, expressed as 60Co equivalent doses, and the effects on cell division following treatment with intracellular and extracellular 111In were measured in Chinese hamster V79 cells. The chromosome aberration yield in cells irradiated by intracellular 111In indicated that damage was induced at a rate of 7.2 X 10(-4) Gy/decay for levels of activity up to 0.075 Bq/cell and 4.5 X 10(4) and 2.9 X 10(4) Gy/decay for intermediate (0.204 Bq/cell) and high (0.389 Bq/cell) levels, respectively. Extracellular 111In-chloride produced damage at a rate of about 6.1 X 10(-12) Gy/decay. As little as 4.4 mBq/cell (about 4.4 X 10(3) Bq/ml of culture) of intracellular 111In was able to affect cell division, whereas extracellular 111In at 1.150 MBq/ml of culture had little effect. These data indicate that the Medical Internal Radiation Dose and International Committee on Radiation Units methods for organ dosimetry may underestimate the potential of intracellular Auger electron emitters to produce radiation damage.

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper No. 5. Genotoxicity tests as predictors of carcinogens: an analysis.

Differences between the results of numerical validation studies comparing in vitro and in vivo genotoxicity tests with the rodent cancer bioassay are leading to the perception that short-term tests predict carcinogenicity only with uncertainty. Consideration of factors such as the pharmacokinetic distribution of chemicals, the systems available for metabolic activation and detoxification, the ability of the active metabolite to move from the site of production to the target DNA, and the potential for expression of the induced lesions, strongly suggests that the disparate sensitivity of the different test systems is a major reason why numerical validation is not more successful. Furthermore, genotoxicity tests should be expected to detect only a subset of carcinogens, namely genotoxic carcinogens, rather than those carcinogens that appear to act by non-genetic mechanisms. Instead of relying primarily on short-term in vitro genotoxicity tests to predict carcinogenic activity, these tests should be used in a manner that emphasizes the accurate determination of mutagenicity or clastogenicity. It must then be determined whether the mutagenic activity is further expressed as carcinogenicity in the appropriate studies using test animals. The prospects for quantitative extrapolation of in vitro or in vivo genotoxicity test results to carcinogenicity requires a much more precise understanding of the critical molecular events in both processes.

Human mutagens: evidence from paternal exposure?

The importance of inherited mutations as a cause of human disease has been established clearly through examples of well-defined genetic anomalies, such as Down syndrome and retinoblastoma. Furthermore, it is suspected that environmental contaminants induce mutations resulting in increased risk for such defects in subsequent generations of persons exposed. The present lack of direct evidence for induced inherited genetic disorders in human beings hampers the development of risk estimation techniques for extrapolation from animal models. The most extensive prospective epidemiologic studies of inherited genetic effects have involved survivors of atomic bomb detonations and patients treated with cancer chemotherapy. In neither case has a significant elevation in inherited genetic effects or cancer been detected in the offspring of exposed individuals. Epidemiologic studies of subjects receiving chronic exposure may be confounded by the effect of maternal exposure during pregnancy. Consideration of only paternal exposure can minimize the confounding influence of teratogenicity, enhancing the resolving power of studies for inherited effects. Using this approach, retrospective (case-control) studies of childhood cancer patients have provided limited but suggestive evidence for inheritance of induced effects. Endpoints, such as congenital malformations and spontaneous abortion following paternal exposure, can also be considered as indicators of heritable mutagenic effects. For example, there is limited evidence suggesting that paternal exposure to anaesthetic gases may cause miscarriage and congenital abnormalities as a result of induced male germ cell mutations. By comparing male-exposure endpoints for which there are human data, as described above, with parallel or similar animal endpoints, such as dominant lethal, inherited cancer and "male teratogenic" effects, it is possible that suitable models for extrapolating to human risk can be developed. In order to establish a clearer relationship between induced mutation and genetic disease, the current surveillance systems should be expanded to include endpoints relevant to genetic study. The relaxation of regulations regarding access to census data could improve the chances of documenting such an association.

Genetic toxicology of phthalate esters: mutagenic and other genotoxic effects.

The effects of DEHP on sperm morphology and on peripheral blood micronuclei were studied for 12 weeks following five subacute IP injections of DEHP at 1/6, 1/12, and 1/60 of the LD50 per day. Sperm morphology was examined in both adult mice and rats, while peripheral blood micronuclei were scored in mice up to 4 weeks after treatment. In mice, DEHP at 1/6 LD50 significantly depressed body weight gain for up to 12 weeks after treatment, and reduced epididymal sperm number by 4 weeks. Numbers of morphologically abnormal sperm did not differ from controls in the 12 weeks following treatment. In addition, DEHP did not increase the numbers of peripheral blood micronuclei. Studies in the rat indicated that exposure to doses of 1/6 and 1/12 of the LD50 per day of DEHP resulted in a reduced gain in body weight compared to controls. Testis weight, sperm number, and numbers of morphologically abnormal sperm were unaffected by DEHP following treatment. In separate experiments, DEHP did not induce sister chromatid exchange (SCE) or DNA damage in Chinese hamster ovary (CHO) cells. Although DEHP is known to cause testicular atrophy in rats and to a lesser extent in mice, it did not cause an increase in abnormal sperm in either species. Together with the CHO and micronucleus data, these findings suggest that DEHP has a low probability of causing genetic damage capable of being transmitted through the male germ line.

Sister chromatid exchange analysis in the colonic and small intestinal epithelium of the mouse.

This report describes a method which produces high quality chromosome preparations from the colon and the small intestine of the mouse. These preparations are suitable for sister chromatid exchange (SCE) analysis because the pretreatment used to dissociate the cells does not prevent sister chromatid differentiation and because very little tissue handling is required at the time of killing, thus allowing sampling from several animals within a short period of time. Since the procedure used to make chromosome preparations from the tissue samples is very simple, no cytogenetic experience is required to make excellent chromosome preparations using this method. To demonstrate the utility of this method, the induction of SCEs by cyclophosphamide was measured in small intestine and colon samples collected from the same animals. The results indicate that this method can be used to determine dose responses in both tissues.

Detection of nuclear anomalies in the colonic epithelium of the mouse.

Colon carcinogens produce a variety of nuclear anomalies including pyknotic, karyorrhectic, and micronucleated cells in the colonic epithelium within a few hr. Two model carcinogens, 1,2-dimethylhydrazine and gamma-rays, have been used to determine appropriate techniques, conditions, and scoring criteria for detecting such nuclear anomalies most efficiently. The results show that a rapid and sensitive assay for nuclear anomalies can be conducted with a variety of preparation techniques. We anticipate that the assay will be useful as a screen for potential colon carcinogens in the diet or elsewhere. The assay as recommended by us requires at least five animals per group and takes about 1 hr to analyze. A single sample at 6 or 24 hr after treatment should detect most carcinogens.

Mechanism of induction of nuclear anomalies by gamma-radiation in the colonic epithelium of the mouse.

The induction of karyorrhexis and "nuclear anomalies" in colonic crypt cells has been correlated positively with the induction of colonic tumors by chemical treatment. These nuclear anomalies occur in the proliferative region of the crypt and exhibit a variety of morphological characteristics. Some nuclear anomalies resemble the micronuclei that arise from chromosomal fragments after mitosis. Here, we report that the nuclear anomalies observed within the first few hr of insult with gamma-radiation are independent of mitosis for their expression, as evidenced by failure of colchicine to inhibit their induction, and do not arise from chromosomal material lost during mitosis.

Transient DNA lesions induced by benzo[a]pyrene in Chinese hamster ovary cells.

Alkaline sucrose gradient sedimentation analysis was used to detect DNA lesions induced by benzo[a]pyrene B(a)P in Chinese hamster ovary cells. The number of lesions detected immediately following treatment with 10(-4) M B(a)P was related directly to the duration of treatment. When treated cells were incubated in a B(a)P-free medium, the majority of lesions disappeared rapidly and could no longer be detected 15 min following treatment. These data indicate that a population of B(a)P-induced DNA lesions may be removed by a rapid DNA-repair process. The transient nature of such lesions should be considered when assays for DNA damage or repair are designed and interpreted.